Background/Purpose:

14-3-3 proteins are ubiquitously expressed intracellular chaperonins. We previously showed that the h isoform is 1) uniquely expressed in the synovial fluid and serum of patients with inflammatory arthritis 2) differentially expressed in established rheumatoid arthritis (RA) and 3) a novel extracellular mediator that may contribute to the disease process. This study examines 14-3-3h serum levels and its pathophysiological role in early disease.

Methods:

Serum 14-3-3h was measured in 37 DMARD-naïve early RA patients, 25 from the Toronto Early Arthritis Cohort (TEACH) and 12 from the intensified-COBRA cohort, and 50 osteoarthritis (OA) controls using an investigational-grade 14-3-3h ELISA. 2-tailed t-tests and Mann-Whitney u-tests were run to compare group differences in serum concentrations. An ROC curve was generated and sero-positivity rates of 14-3-3h, rheumatoid factor (RF) and anti-citrullinated cyclic peptide (anti-CCP) were evaluated. To examine the effects of 14-3-3h on intracellular signalling in monocytes, THP-1 monocytic cells were stimulated with 12.5ng/ml of recombinant human 14-3-3h (030min) and activation of the MAPK signalling cascades (ERK, JNK/SAPK and p38) were assessed by immunoblot analysis using phosphospecific antibodies. The mRNA levels of IL-1b, IL-8, CCL2/MCP-1 and CCL4/MIP1- b following 18h incubation with a dose range of 0.10 to 100ng/ml of recombinant human 14-3-3h or vehicle were assessed by RT-PCR. Densitometry was used to measure % change with stimulation above control.

Results:

Mean and median 14-3-3h serum concentrations in RA patients [TEACH (3.13 & 0.63ng/ml) and i-COBRA (5.90 & 1.43ng/ml)] were significantly higher than in OA controls (0.32 & < 0.20ng/ml), p-values <0.0006 and <0.0001, respectively. The corresponding areas under the ROC curve were 0.72 (95%CI 0.580.86) and 0.87 (95%CI 0.721.00) for TEACH and i-COBRA RA patients versus OA. 14-3-3h, RF and anti-CCP positivity were 60%, 32% and 44% in TEACH and 82%, 82% and 82% in i-COBRA. 72% of TEACH and 100% of i-COBRA RA patients were positive for any one of the three markers. Stimulation of THP-1 cells with 14-3-3h activated ERK and JNK/SAPK by 227% and 87% above control at 5 and 2 min, respectively. No activation of p38MAPK was observed at any of the time points. 14-3-3h was associated with potent induction of transcripts of early pro-inflammatory factors, with IL-8 (47% increase at 0.1ng/ml) and MIP-1b (53% increase at 0.25ng/ml) being the most sensitive followed by MCP-1 (44% increase at 0.5ng/ml) and IL-1b (49% increase at 5ng/ml).

Conclusion:

14-3-3h is differentially expressed in the serum of patients with early RA compared to OA controls and is a novel factor that may contribute to pathological processes involved in early disease. 14-3-3h serum expression when combined with standard serological RA tests may mark early RA.